Download RNA Polymerase and Associated Factors Part C by Adhya S. (ed.), Garges S. PDF

By Adhya S. (ed.), Garges S.

RNA polymerase is molecule vital to gene transcription. in addition to linked elements, RNA polymerase is a part of the method during which RNA is transcribed to provide a protein.* building and purification of RNA polymerases* DNA microarrays and bacterial gene expression* sensible research of transcription components

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Chem. 351, 221 (1970). 18 RNA polymerase structure and properties [2] buffer at a flow rate of ca. 25 ml/h. 31 RNAPcontaining fractions are dialyzed into RNAP storage buffer [TMED, 50%  (v/v) glycerol, 100 mM NaCl] and stored at À20 . These fractions retain activity for at least several years. At this stage, the purified RNAP contains all the core subunits (including ) and a substoichiometric complement of A. Other  factors are also present at much lower levels. 27 Separation of EA from core (E containing a small amount of ED) can be achieved using heparin–agarose chromatography and a shallow gradient7 or using FPLC MonoS chromatography (see later).

Lewis, and M. D. Yudkin, J. Bacteriol. 184, 5583 (2002). 45 T. Magnin, M. Lord, and M. D. Yudkin, J. Bacteriol. 179, 3922 (1997). 46 E. A. Campbell and S. A. Darst, J. Mol. Biol. 300, 17 (2000). 22 RNA polymerase structure and properties [2] Purification of A A is purified from E. 4% glucose and 100 g/ml ampicillin. 4 mM and growth continued for 2 h. 2 ml of lysis buffer 3 (lysis buffer 1 without MgCl2), hen egg white lysozyme is added to 130 g/ml, and the suspension is incubated on ice for 10 min.

Helmann, J. Mol. Biol. 249, 743 (1995). 32 [2] B. 1 mM DTT, 5% glycerol, 1 mM PMSF), and lysed by three passages through a French pressure cell at 20,000 psi. 1 M NaCl. 0 M NaCl. RNAP-containing fractions are pooled and precipitated with ammonium sulfate (65% saturation), and the precipitate is collected by centrifugation (17,000 g). 5 M NaCl. The peak fractions are pooled, diluted threefold with TMED with 5% glycerol, and loaded on to an FPLC Mono Q HR5/5 column. 1 mM DTT, 50% glycerol) and stored at À20 .

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